1. Field of the Invention
This invention relates to methods and compositions for separating and isolating biological materials by means of magnetic labelling. The material to be labelled is reversibly anchored to a support before being labelled. The magnetic labels contemplated are small enough to be appropriate for use in separating and isolating biological material in the microscopic range, e.g. chromosomes, mitochondria, chloroplasts, and Golgi apparatus. Binding compositions such as nucleic acid probes or antibodies form part of the labelling complex. After release from the support, the labelled material is isolated by sorting in a magnetic field.
2. Description of the Related Art
Biological material is a stew of heterogeneous ingredients, which needs to be separated into component parts prior to many investigations and procedures. The separation methods vary depending on the absolute and relative sizes of the material to be separated, and the degree of purity which must be achieved. To separate relatively large materials, that is, the "meat" of the stew, centrifugation, filtering and density gradient sedimentation are some of the relatively crude methods that are appropriate. To separate small, minute cellular components, the "spices", e.g., chromosomes, flow cytometry has been a major method. Some materials, e.g., the Golgi apparatus, have not been satisfactorily separated by any means as an individual, intact cellular component.
Although there are many variations on the flow cytometry theme, the basic principle of this method is to label the cellular material, for example, chromosomes, according to its DNA content, which will be generally correlated with size, and to separate the material into collecting tubes by laser beams that quantitatively measure the DNA content. Flow separation of human chromosomes has been somewhat successful in completely separating some of the 23 pairs of chromosomes but has resulted in some aggregation of chromosomes of similar sizes, e.g., the human chromosomes 21-22 and Y. Therefore, this method is of limited usefulness. Present methods for separation of small biological materials, e.g., chromosomes, which are in the range of 0.2 to 10 microns or even smaller entities such as B-chromosomes, minichromosomes and double-minute chromosomes, are inadequate or unavailable. Flow cytometry is not sensitive enough to guarantee separation of small individual components which often sort with the debris. Somatic cell methods (hybridization, microcell fusion) can isolate chromosomes but the methods are laborious and unpredictable. What is needed is some kind of separation method that is not solely dependent on size differences, but is related to other inherent properties of the materials to be separated.
A. Chromosome Isolation and Sorting
Cellular components which are major targets for methods of separation and isolation are chromosomes. It is important to be able to analyze the chromosomal composition of a cell, cell line, tissue or organism, because deviations from the correct number of chromosomes usually produce phenotypic abnormalities. The phenotype results from the interaction of the genetic complement and the environment. Structural chromosomal aberrations may also produce abnormalities, if the genetic balance is disrupted. Chromosomal aberrations may be detected by standard karyotyping, wherein photomicrographs of the chromosomes in individual cells are analyzed. Another method of detection is to sort chromosomes from large numbers of cells, by flow cytometry (Gray, 1990) and to compare the results with a standard pattern.
For other purposes, it is also desirable to isolate groups of identical chromosomes. For example, in humans, aneuploidy of chromosome 21.sup.1 is responsible for Down syndrome. To study the properties of this clinically important chromosome, it is helpful to separate large numbers of No. 21 chromosomes from the others, or to isolate individual No. 21's in a background of chromosomes from another species. The former may also be achieved by flow cytometry, the latter by somatic cell hybridization techniques, for example. Although somewhat useful, current techniques for chromosome isolation and sorting each have serious limitations in terms of time and cost, unpredictability, inaccuracies due to contamination, and destruction or alteration of the chromosomes during processing. FNT .sup.1 Chromosomes are conventionally numbered, usually in decreasing order according to size as determined microscopically at metaphase.
B. Flow Cytometry
Flow cytometry techniques are used for both analysis and sorting of biological macromolecules and cells (Darzynkiewicz and Crissman, 1990).
Flow cytometry currently is the primary method for the purification of specific chromosomes. However, its efficacy is limited by the amount of time (hours or even days) required to sort large quantities of a single chromosome. Furthermore, it is not yet possible to reliably separate different chromosomes whose DNA contents are similar. There is some cross-contamination of chromosomes having similar sizes. Furthermore, it is impossible to separate individual chromosomes by DNA content alone in species such as the mouse whose karyotype consists of similarly sized telocentric chromosomes (these are chromosomes with their primary constrictions, the centromeres, located at one end). In such cases, either somatic cell hybridization, a method whereby a given chromosome may be laboriously isolated in a genetically different background by cell fusion and selection, or a cell type with a non-uniform karyotype, such as is found in mouse strains carrying single Robertsonian translocation chromosomes, which are of a different size and arm-ratio from the nontranslocated chromosomes, may be employed.
The ability to isolate and sort specific chromosomes is of use in the study of both normal and malignant cell processes, and is an essential first step in the creation of chromosome-specific libraries for cloning. For instance, flow sorting of specific chromosomes has been used to detect deletions in apparently balanced translocation chromosomes (Cooke, et al., 1989). Because the deleted translocation is not the quantitative sum of its component parts, the missing part may be deduced. Flow cytometry has also been used to investigate genetic changes associated with the malignant state, as exemplified in a case of familial renal carcinoma, where two oncogenes have been translocated to the derivative chromosomes of a cancer-related translocation (Harris, et al., 1986), a change that is detectable quantitatively.
Although some success has occurred with previous isolation and sorting methods, these are still laborious and inaccurate. Finally, debris and cross-contamination in flow-sorted preparations compromise the utility of this approach. Thus, a method to reliably generate single chromosome preparations in a short time would have many applications in library construction, cloning, and the analysis of genetic changes such as those that occur in cancer.
C. In-Situ Hybridization
In-situ hybridization of middle or high repeat DNA sequences using radioactive probes has been available to those skilled in the art for a long time (Pardue and Gall, 1970). More recently, labelling with non-radioactive probes has been favored to detect the location of the hybridized probe. Hybridization occurs between complementary nucleic acid sequences if conditions are appropriate. As is well known to those skilled in the art, the hybridization conditions, i.e., "stringency," may be controlled to permit hybridizing between segments of various per cent complementarity. Hybridization may occur between probes and segments of different sizes, for example, from high or middle repeat sequences, to single copy DNA. Hybridization may occur between probes and any cellular component containing nucleic acid. Chromosome-specific, repetitive sequence hybridization probes exist (Moyzis, et al., 1987).
Fluorescent rather than radioactive probes are also available, but there are problems in the detection of the fluorescent signal for single-copy DNA sequences because of the weak signal from a small target. Methods for amplification of the signal have been explored, e.g. by enhancing the strength of the signal itself, and are well known to those skilled in the art. Enhancing the signal detection (e.g., by digital image enhancement, Viegas-Pequignot, et al., 1989) is another approach.
There have been attempts to conduct in-situ hybridizations on whole chromosomes in suspension. Success in this venture would facilitate the Fluorescence Activated Cell Sorter (FACS) process for chromosome sorting.
Manning, et al. (1975) reported a scanning electron microscope in-situ hybridization method based on avidin-polymer spheres binding to biotin-coupled nucleic acid probes hybridized to polytene chromosomes.
Dudin, et al., (1988) carried out in-situ hybridization on suspensions of chromosomes prepared as for flow cytometry. Human genomic DNA biotinylated by nick translation was used to label human chromosomes by in-situ hybridization in suspension. The authors stated that streptavidin was covalently coupled to the surface of magnetic beads and these were incubated with the hybridized chromosomes. The human chromosomes from Chinese hamster X human hybrid cell lines were bound to the magnetic beads through the biotin-streptavidin complex and separated from non-labelled Chinese hamster chromosomes by a simple permanent magnet. Hybridization was visualized by additional binding of avidin-FITC (fluorescein) to the unoccupied biotinylated human DNA bound to the human chromosomes. Large magnetic beads (4 .mu.m) were used in these experiments.
These authors noted that for high purity sorting of chromosomes, the heterogeneous aggregates, in this case hamster mixed with human chromosomes, and interphase nuclei, must be significantly reduced or eliminated. They suggested 1 g sedimentation preferably prior to magnetic separation as a means of solving these problems. Another problem these investigators encountered was severe clumping when large numbers of chromosomes/magnetic beads were used. Overall, this approach has not been very successful due to problems with adventitiously adsorbed contaminants, and chromosome aggregation and loss in the centrifugation steps required for changing solutions during the procedure. Furthermore, buffer components such as hexylene glycol used in preparing chromosome suspensions cause excessive condensation of the chromosome and consequently a loss of accessibility of sequences within the structure of the condensed chromosome. This reduction in sensitivity is obviously detrimental to sequence detection. In addition, the large magnetic particles used which are about the size of some chromosomes in metaphase (.about.3-4.mu.) reduce yield because there is a low efficiency of labelling, probably due to limited target accessibility. Finally, lack of reproducibility means that suspension in-situ hybridization cannot be a solution to the problems of flow sorting of chromosomes.
D. Use of Magnetic Systems
Large paramagnetic particles are currently being used in conjunction with DNA diagnostics and cell separations (Kvalhelm, et al., 1987). Magnetic affinity chromatography has also recently become a viable alternative method of purifying biological structures (Menz, et al., 1986). Magnetic solid supports with specific affinity couples are used for separating cells, cell organelles, and microorganisms (see introduction in Dudin, et al., 1988). One member of the affinity couple is usually an antibody covalently bound or physically absorbed to magnetic microspheres. Some of those used are polystyrene beads containing ferric oxide (Fe.sub.3 O.sub.4) particles (see reviews by Lea, et al., 1985; Howell, et al., 1988).
Magnetic beads were originally developed for immunoassays. They have also been used to separate DNA and RNA (Uhlen, 1989). Some of the original magnetic particles, made by the polymerization of acrylamide and agarose with paramagnetic materials, were heterogeneous in size and magnetic content. Hydrophilic beads have now been developed that are more homogenous in size, density and amount of magnetized material. Such beads sediment homogeneously in magnetic fields. The chemical structure of the particle surface may be varied, providing a flexible system for the attachment of biomolecules.
A magnetic cell sorting system for separation of large numbers of cells according to specific cell surface markers was reported by Miltenyi, et al. (1990). Cells were labelled sequentially with biotinylated antibodies, fluorochrome-conjugated avidin, and superparamagnetic biotinylated microparticles. These cells were then separated on high gradient magnetic (HGM) columns. Retained cells were then eluted from the column. This method was said to be compatible with analysis of separated cells by fluorescent microscopy or flow cytometry (FACS). Miltenyi, et al. (1990) have implemented a suggestion by Molday and Molday to combine small superparamagnetic microparticles and high gradient magnetic (HGM) fields to separate cells; they call this MACS.
E. Reversible Immobilization
Recently, various methods have been developed for the immobilization of biological structures. These have found numerous applications in molecular biology, for instance. Biologically active structures such as enzymes have immobilized on matrices such as silica for use in studies of biochemical catalysis (Wu and Walters, 1988). Oligonucleotides are currently synthesized on controlled pore glass supports (Damha, et al., 1990). Various purification methods and DNA capture methods rely on the immobilization of molecules of interest on a solid support (Bebee and Gebeyehu, 1990). Biosensors and monitoring systems have also been designed using immobilized biomolecules (Lehman, 1990).
Immobilization methods can utilize a variety of chemical crosslinking agents (Staros, 1982), both cleavable and non-cleavable. Thus, immobilization can be reversible or not, as required. However, these methods have not been applied to isolating and sorting of small biological materials. The combination of methods in the present invention addresses the separation and isolation of small biological materials.